Mass Spectrometric Screening for Improving Enzymatic Conversion of Formaldehyde into C2 and C3 Products.

One-carbon biomanufacturing offers a sustainable route for producing value-added chemicals. Glycolaldehyde synthase (GALS), an engineered enzyme from Pseudomonas putida, catalyzes a key step by condensing formaldehyde (FALD) into carbohydrate molecules, such as glycolaldehyde (GALD) and dihydroxyacetone (DHA). However, its industrial application is limited by low catalytic efficiency and lack of high-throughput screening methods. Here, we developed a mass spectrometry (MS)-based assay for simultaneous detection and quantification of GALD and DHA products from whole-cell FALD biotransformation by GALS-expressing Escherichia coli. Integrating this MS assay with a robotic biofoundry, we created and screened site-directed mutagenesis libraries targeting seven key residues of GALS, achieving a throughput of ∼10 s per sample. Several improved mutants were successfully isolated, including one with a 3.7-fold increase in kcat for GALD production and another with a 5-fold reduction in Km for DHA production, compared to the wild type GALS. Molecular dynamics simulations were applied to understand the mutational impact on substrate binding and product specificity. This high-throughput workflow may be extended to engineer other enzymes for one-carbon feedstock utilization.